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Abstract

Background Nonrheumatic stenosis of trileaflet aortic valves, in which calcification is a prominent feature, has been termed a “degenerative” condition, but it has been demonstrated recently that chronic inflammation is a characteristic feature of the developing lesion of aortic stenosis. This observation raised the possibility that calcification in the aortic valve might be actively regulated. Thus, the present study investigated whether osteopontin, a protein implicated in the regulation of both normal and dystrophic calcification, could be detected in lesions of valvular aortic stenosis.

Methods and Results Morphological and immunohistochemical studies were performed on 14 human aortic valves, representing a range of pathology from normal to clinically stenotic. The extent of calcification and macrophage accumulation and their relation to the presence of osteopontin protein were characterized. Highly statistically significant associations were found between the degree of osteopontin expression and the degrees of both calcification and macrophage accumulation in early through late lesions of aortic stenosis. Further, in situ hybridization localized osteopontin mRNA to a subset of lesion macrophages.

Conclusions These results suggest that, rather than representing a degenerative and unmodifiable process, calcification in aortic stenosis may be, in part, an actively regulated process with the potential for control either through modification of inflammation or synthesis of proteins such as osteopontin, which may modulate calcification in this tissue.

Nonrheumatic aortic valvular stenosis is a common disease of the elderly, in whom the estimated prevalence of critical aortic stenosis is 2.9%.1 Stenosis of a trileaflet aortic valve has been termed a “degenerative” condition,2345 but recent studies have demonstrated that the developing lesion of aortic stenosis has many components of a chronic inflammatory process, including accumulation of both macrophages and lymphocytes.6 Further, calcification is prominent in valvular aortic stenosis and has important functional consequences.2345 Recognition of an association of inflammatory cell accumulation with developing aortic valve lesions led us to investigate the possibility that protein mediators of calcification might be present in aortic valve leaflets.

One protein thought to play a role in calcification is osteopontin. Osteopontin is a highly acidic, 44-kD glycoprotein789 and is one of a group of noncollagenous matrix proteins of bone.910111213 It also has been implicated in cell adhesion and spreading14 and in cell cycling.15 Osteopontin binds readily to hydroxyapatite10 and may mediate adherence of osteoblasts and osteoclasts to bone matrix through an arginine-glycine-aspartate (RGD) integrin-binding sequence.7 Osteopontin also has been implicated in dystrophic calcification; specifically, the protein is present in renal stones,16 and its secretion by renal distal tubules is upregulated in a rat model of nephrolithiasis.17 However, there is controversy as to whether osteopontin promotes1617 or inhibits18 renal stone formation. Further, several studies recently have demonstrated associations of osteopontin with both calcification and atherosclerosis in human arteries.1920212223 In contrast, arterial levels of osteonectin, another noncollagenous bone matrix protein, have been shown to decrease as atherosclerosis and calcification developed.19

Therefore, demonstration of an association between the presence of osteopontin and calcification in human aortic valves would be consistent with the hypothesis that calcification in this tissue is, at least in part, actively mediated rather than a merely passive, or degenerative,2345 phenomenon. The present study was undertaken to address the following specific questions: (1) Is osteopontin preferentially present in diseased compared with normal aortic valves? (2) If so, is osteopontin associated with calcification or inflammatory cell accumulation? and (3) Which cells produce this protein?

Methods

Human Aortic Valvular Tissue

Human aortic valvular tissue used for morphological and histochemical analysis was 11 valves obtained at autopsy from patients without clinical aortic stenosis and 3 valves obtained at surgery from patients undergoing valve replacement for clinical aortic stenosis. The valve leaflets were fixed in methanol Carnoy’s fixative (60% methanol, 30% chloroform, 10% acetic acid) and embedded in paraffin. Sections of the leaflets were stained with hematoxylin-eosin or with Verhoeff–van Gieson’s stain for valve morphology and with von Kossa’s stain to identify calcium phosphate mineral.

For in situ hybridization studies, an additional 9 leaflets were obtained from the native hearts of patients undergoing cardiac transplantation for ischemic or idiopathic cardiomyopathy and were fixed in formalin within 2 hours of organ removal to minimize ex vivo mRNA degradation. Although none of these patients had either clinically evident or echocardiographically detectable aortic stenosis, some of the leaflets contained early or moderate aortic valvular lesions.6

Immunohistochemical Reagents: Antibodies and Antisera

Immunohistochemistry was performed with the following antibodies: (1) LF7, a rabbit polyclonal anti-osteopontin antiserum raised against human bone–derived osteopontin12 and used previously for immunohistochemistry on human arterial tissue,2023 was a kind gift of Dr Larry Fisher and was used at a titer of 1:1000 to localize osteopontin protein20 ; (2) mouse monoclonal antibody anti-CD68,24 used at a titer of 1:1000 to identify macrophages; and (3) mouse monoclonal antibody anti–smooth muscle α-actin,25 used at a titer of 1:1000 to identify cells with contractile proteins, which in the context of aortic valvular tissue might represent either myofibroblasts or smooth muscle cells (SMCs).

Immunohistochemistry

Single-label immunoperoxidase staining of 6-μm aortic valve sections was performed as described previously.2627 Briefly, tissue sections were deparaffinized with xylene and then rehydrated with graded alcohols. The slides were blocked with 3% H2O2, washed with PBS, incubated for 30 minutes with the primary antiserum or antibody, and then washed again with PBS. A biotin-labeled secondary antibody, either anti-rabbit (for LF7) or anti-mouse (for anti–smooth muscle α-actin or anti-CD68), then was applied for 30 minutes, followed by an avidin-biotin-peroxidase conjugate (ABC Elite; Vector Laboratories) for 30 minutes. 3,3′-Diaminobenzidine with nickel chloride was used as a chromogen, yielding a black reaction product. Cell nuclei were counterstained with methyl green. On formalin-fixed tissue, nickel chloride was omitted from the 3,3′-diaminobenzidine chromogen reaction, thus yielding a brown reaction product, and sections were counterstained with hematoxylin.

Negative controls included substitution of primary antiserum/antibody with PBS or with either nonimmune rabbit serum or irrelevant antibodies, as appropriate, at the same titer.

Statistical Analysis

The statistical validity of the apparent associations between osteopontin and calcification and between osteopontin and macrophage accumulation was evaluated in the following fashion. The leaflet from each of the 14 patients was divided into three segments: the base, the midportion, and the tip; each segment was then evaluated for osteopontin, macrophage accumulation, and calcification. Each of these characteristics was graded on a semiquantitative scale, ranging from 0, which represented none or normal, up to 4, which represented the most widespread distribution of the characteristic. Associations between osteopontin and calcification and between osteopontin and macrophage accumulation then were examined with Mantel-Haenszel’s test for linear association with spss/pc+ statistical software. Significance was set at the P<.05 level.

Riboprobe Preparation

A 1.5-kb human osteopontin cDNA (clone OP10) cloned into the vector pBluescript SK (fl−) (Stratagene) was a kind gift of Dr Larry Fisher, National Institutes of Health. The plasmid was linearized with Xba I for antisense riboprobe transcription or with Xho I for sense (control) riboprobe transcription. Reagents for riboprobe synthesis were obtained from Promega, except for 35S-UTP (1000 to 3000 Ci/mmol), which was obtained from New England Nuclear.

The riboprobe transcription reaction mixtures contained 1 μg cDNA; 250 μCi 35S-UTP; 500 μmol/L each of rATP, rCTP, and rGTP; 40 U RNasin (Promega); 10 mmol/L DTT; 40 mmol/L Tris; and 10 U of either T7 polymerase (for antisense transcription) or T3 polymerase (for sense transcription). After 60 to 75 minutes of incubation at 37°C, the cDNA was digested by addition of 1 U RQ1 DNase (Promega), and incubation was continued for an additional 15 minutes at 37°C. Free nucleotides were separated in a Sephadex G-50 column, and the riboprobes were used within 24 hours of synthesis.

Results

Immunohistochemistry

Association of Osteopontin With Calcification and Macrophage Accumulation

On the 14 valve leaflets fixed in methanol Carnoy’s solution, two general patterns of calcification could be seen. The most common pattern of osteopontin protein expression was an association of osteopontin with both calcification and macrophage accumulation, as demonstrated in Fig 1⇓. These photomicrographs were obtained at the junction of the midportion of the leaflet and the leaflet tip. The aortic surface of the leaflet is located at the top of the sections. This developing aortic valvular lesion is characterized by displacement and fragmentation of the basement membrane and accumulation of lipid, protein, and cells. Comparison of Figs 1A⇓ and 1B⇓ demonstrates that in the lower left portion of each section, black areas of calcium mineral deposition (Fig 1A⇓) are surrounded by osteopontin protein (Fig 1B⇓). In this region of the lesion, macrophages also could be found (Fig 1C⇓). Fig 1B⇓ demonstrates that much of the osteopontin protein appears to be extracellular. However, occasional cells in closest proximity to the bulk of the osteopontin staining have intracellular staining for the osteopontin protein and, by comparison with Fig 1C⇓, appear to be macrophages. In contrast, other subsets of macrophages located near the surface of the leaflet and below the region of calcification at the junction between the fibrosa and ventricularis are negative for the osteopontin protein. These findings raise the possibility that a subset of lesion macrophages is actively producing and secreting osteopontin to modulate extracellular calcification in this area. Staining with the antibody against α-actin was negative in this region (data not shown). This pattern of osteopontin expression in association with calcification and macrophage accumulation was present in leaflets from 9 of 14 patients.

Association of osteopontin protein and calcification with macrophage accumulation. Views of the central portion of a moderate lesion of aortic stenosis (A through C). The von Kossa stain demonstrates a region of extracellular calcification (A, black staining, left) in which extracellular osteopontin protein is detected with the LF-7 antibody (B, black immunoreaction product, short black arrows, left). Intracellular osteopontin protein also is detected (B, long black arrows, central region), and these cells are identified with the anti-CD68 antibody as both non–foam cell and foam cell macrophages (C, black immunoreaction product). However, other macrophages do not contain osteopontin protein, as seen both at the surface of the lesion (B, open arrows, top) and at the junction of the fibrosa and ventricularis (C, bottom). Magnification ×200; von Kossa’s stain (A) or methyl green counterstain (B and C).

Association of Osteopontin With Calcification

The second pattern of osteopontin expression, shown in Fig 2⇓, was of focal areas of calcification seen in association with osteopontin deposition in areas without inflammatory cell infiltrate. The Verhoeff–van Gieson (Fig 2A⇓) and hematoxylin-eosin (Fig 2B⇓) stains demonstrate a region of a valve leaflet with mild thickening of the fibrosa (upper layer of the valve leaflet). The aortic surface of the valve is at the top of the leaflet, with the ventricular surface at the bottom. The leaflet consists of three layers: the collagen-rich fibrosa (upper layer, Fig 2A⇓ through 2F), the spongiosa (central layer), and the elastin-rich ventricularis (bottom layer, Fig 2A⇓ through 2F). In this leaflet, there is an association between osteopontin protein (black immunoreaction product of the anti-osteopontin antiserum, Fig 2C⇓) and macroscopic calcification (black staining with von Kossa’s stain, Fig 2D⇓). Only scattered macrophages are present in the ventricularis and in the spongiosa (black reaction product of the anti-CD68 anti-macrophage antibody, Fig 2E⇓), but no macrophages are present in the area with osteopontin and calcification. The absence of SMCs or myofibroblasts from the regions of calcification is demonstrated by the absence of staining in the fibrosa with an antibody against α-actin (Fig 2F⇓). This association of calcification with osteopontin protein, occurring in the absence of macrophage accumulation, was uncommon, being present in leaflets from 2 of 14 patients. Three additional patients had neither microscopic nor macroscopic calcification nor immunohistochemically detectable osteopontin protein. Thus, all 11 valve leaflets with calcification contained osteopontin protein, whereas 9 of the 11 also contained macrophages in regions with osteopontin protein.

Association of osteopontin protein with calcification. The leaflet sections shown are oriented with the upper layer (fibrosa) at the top and the bottom layer (ventricularis) at the bottom. Valve morphology is shown with Verhoeff–van Gieson’s (A) and hematoxylin-eosin (B) stains. The hematoxylin-eosin stain (B) discloses an area with basophilic extracellular staining in the fibrosa in which osteopontin protein is detected with the LF-7 antiserum (C, black immunoreaction product) in association with calcification, as demonstrated with von Kossa’s stain (D, black staining). Black arrows indicate areas of calcification in B through D. Although the anti-CD68 antibody detects scattered macrophages (E, black immunoreaction product) present in the ventricularis and in the central layer, or spongiosa, of the leaflet, none of this inflammatory cell type is present in the region with osteopontin protein and calcification. Likewise, the anti–α-actin antibody (F, black immunoreaction product) does not detect α-actin–containing cells in the region of calcification, although some are present in the ventricularis. Only 2 of 14 leaflets contained this combination of osteopontin protein and calcification without associated inflammatory cells. Magnification ×100 (A through F); Verhoeff–van Gieson’s stain (A), hematoxylin-eosin counterstain (B), von Kossa’s stain (C), or methyl green counterstain (C through F).

Statistical Analysis of Immunohistochemistry Findings

Mantel-Haenszel’s tests for linear association were performed for each of the three anatomic regions of the leaflets for the comparisons of the semiquantitative assessments of osteopontin expression and calcification and of the semiquantitative assessments of osteopontin expression and macrophage accumulation, as described in “Methods.” The Table⇓ lists the Mantel-Haenszel test scores at each of the three anatomic regions of the leaflets, namely, base, midportion, and tip, for the comparison of osteopontin versus calcification and osteopontin versus macrophage accumulation. The Mantel-Haenszel scores indicate highly statistically significant associations between the amount of osteopontin and the degree of calcification for each of the three regions of the leaflet. Likewise, the scores also indicate highly significant associations between the amount of osteopontin and the amount of macrophage accumulation.

Association of Osteopontin With Calcification and Macrophage Accumulation

In Situ Hybridization

To determine which cell types in the lesion synthesize osteopontin, in situ hybridization was performed on formalin-fixed aortic valve leaflets obtained from the excised native hearts removed from nine patients at the time of cardiac transplantation. Aortic valvular tissue from these patients had been fixed in formalin within 2 hours of organ removal, thus maximizing mRNA preservation for in situ hybridization studies. Fig 3⇓ demonstrates the presence of osteopontin mRNA and protein in one of the valve lesions examined. In situ hybridization with the 35S-labeled antisense osteopontin riboprobe (Fig 3A⇓) demonstrates hybridization in mononuclear cells in a developing valve lesion, whereas no specific hybridization is seen on an adjacent section with the sense (control) riboprobe (Fig 3B⇓). Staining of an adjacent section with the CD68 antibody (Fig 3C⇓) demonstrates that these cells represent a subset of macrophages, thus confirming that a subset of lesion macrophages can synthesize osteopontin. The presence of osteopontin protein in these cells is confirmed with the LF7 antiserum (Fig 3D⇓). Staining of neighboring sections with the α-actin antibody and a CD3 antiserum6 confirmed the absence of SMCs and T lymphocytes from this osteopontin mRNA- and protein-containing region (data not shown). Scattered extracellular staining for osteopontin is present in the region adjacent to these osteopontin mRNA-positive macrophages (short black arrows, Fig 3D⇓). Expression of osteopontin mRNA by other cell types in the valves, eg, fibroblasts or SMCs, was not detected on these specimens with this technique. Further, the occasional macrophages typically present in the ventricularis of nondiseased valves6 did not contain osteopontin mRNA or protein.

In situ hybridization localizes osteopontin mRNA to a subset of lesion macrophages. Hybridization with a 35S-labeled antisense riboprobe demonstrates the presence of osteopontin mRNA (A, black autoradiography grains, left) in cells in an aortic valve lesion, whereas hybridization with the sense (control) riboprobe demonstrates no specific hybridization (B). The anti-CD68 antibody identifies these cells as macrophages (C, black immunoreaction product, left), which also contain osteopontin protein, detected with the LF-7 antiserum (D, long black arrows, right). Extracellular osteopontin also is present in the region adjacent to these macrophages (D, short black arrows, right). Note that a brown chromogen was used in these slides, so the osteopontin immunoreaction product is less obvious than in Figs 1C⇑ and 2C⇑. Magnification ×400; hematoxylin-eosin stains (A and B) or hematoxylin only (C and D).

Discussion

Nonrheumatic valvular aortic stenosis is a common disease of the elderly, and as such has been termed a degenerative condition,2345 even though the majority of elderly individuals do not have clinically significant aortic stenosis.1 In valvular aortic stenosis, there is prominent deposition of calcium, which plays a major role in limiting valve mobility.2345 The recent demonstration that the developing lesion of aortic stenosis contains chronic inflammatory cells6 raised the possibility that inflammatory cells modulate calcium deposition in aortic valvular tissue. This study is the first to demonstrate the presence and active synthesis in aortic valvular tissue of a protein, osteopontin, implicated in both normal7813 and dystrophic calcification161720212223 in a variety of other tissues. Further, the study demonstrates significant associations between the degree of osteopontin present and the degrees of calcification and macrophage accumulation. Finally, in situ hybridization studies demonstrate that a subset of macrophages in aortic valvular tissue may actively synthesize the osteopontin protein.

Several different proteins have been implicated in calcium deposition in bone and other tissues including, but not limited to, bone morphogenetic protein-2a (BMP-2a),28 matrix Gla protein,22 osteonectin,18 and osteopontin.1617181920212223 These proteins have been studied in a disease with chronic inflammation that has several similarities to valvular aortic stenosis, namely, atherosclerosis. Boström et al28 used in situ hybridization to detect BMP-2a mRNA in calcified areas of three human carotid endarterectomy specimens. The cell types expressing BMP-2a were not directly identified on tissue sections, but the authors were able to culture a subset of vascular cells that had several characteristics of vascular pericytes and that could be shown to express BMP-2a mRNA in vitro.28 These authors subsequently showed that these “calcifying vascular cells” also express osteopontin.29 Also, matrix Gla protein, another bone-associated protein, has been detected in human atherosclerotic plaques.22 The observation that BMP-2a, matrix Gla protein, and osteopontin are present both in bone and in calcified vascular tissue suggests that bone and vascular calcification may be very similar processes. In contrast, expression of osteonectin, another protein implicated in calcium deposition, has been shown by Northern blotting and by in situ hybridization to be inversely correlated with atherosclerosis and calcification in human atherosclerotic tissue,18 suggesting that there may be some differences in the regulation of calcification in bone compared with vascular tissue.

Osteopontin has been studied in a variety of normal and pathological circumstances. In bone, osteopontin may be secreted by both osteoblasts and osteoclasts but is invariably present at the forming surface of bone. Further, osteopontin has been posited to play a role in mineral deposition, since it has a high affinity for hydroxyapatite, the major form of calcium and phosphate in bone,7 artery wall,28 and aortic valves.30 The observation that osteopontin is made by osteoclasts has raised the possibility that the protein also plays a role in bone resorption.3132 Also, osteopontin is synthesized in a variety of tissues in which calcium deposition does not occur33 ; therefore, the protein may have other as yet undetermined roles.

However, osteopontin is unique among the noncollagenous matrix proteins of bone for the frequency with which it has been identified in conditions with pathological or dystrophic calcium deposition, such as nephrolithiasis1617 and atherosclerosis.1920212223 The finding that osteopontin is present in aortic valvular lesions and is associated with calcification suggests that calcium deposition in this tissue is, at least in part, an actively regulated and therefore potentially modifiable process. However, these results do not determine whether the effect of osteopontin is to mediate calcium deposition or removal. For example, whereas some authors have suggested that osteopontin mediates renal stone formation,1617 another has suggested an inhibitory role for osteopontin in nephrolithiasis.18 Further, the demonstration that osteopontin is actively synthesized in aortic valve lesions raises the possibility that, as in bone, other proteins implicated in the regulation of calcium deposition111229 may be involved in aortic valve mineralization. The possibility that proteins implicated in bone formation may play a role in aortic valve calcification is further supported by the observations that histologically identifiable bone is present in some aortic valves subjected to balloon valvuloplasty34 and that human aortic valve calcification contains spheroidal particles similar to calcium phosphate particles in bone.35 In addition, lipid deposition36 and abnormal calcium homeostasis37 may play important roles in aortic valve calcification.

This study demonstrates that a subset of valve macrophages actively synthesize osteopontin protein in aortic valvular tissue. Other studies similarly have demonstrated that macrophages may synthesize osteopontin mRNA1922 and contain osteopontin protein22 in human atherosclerotic plaques; therefore, detection of osteopontin in macrophages has precedent. It also has been shown by immunohistochemistry202223 and by in situ hybridization2021 that osteopontin protein can be detected on vascular SMCs. The present study was not able to demonstrate osteopontin expression by another cell type of mesenchymal origin, ie, valve fibroblasts, which may express actin in areas of aortic valvular lesion formation.6 However, because SMCs are not a typical component of aortic valvular lesions, the lack of osteopontin expression by fibroblasts in the present study is not directly comparable. Finally, because circulating osteopontin levels are low, the present study suggests that macrophages may be the primary source of osteopontin protein in valve tissue. However, it should be emphasized that osteopontin expression does not appear to be a basal or normal function of macrophages in this tissue, as evidenced by the lack of expression of the protein by this cell type either in nondiseased aortic valves or in nonlesioned areas of diseased aortic valves, both of which frequently contain resident macrophages.6 This suggests that, in aortic valves, osteopontin expression by macrophages is inducible rather than constitutive and pathological rather than normal.

It is interesting to consider why, although the lesions of both aortic stenosis and atherosclerosis have inflammatory components, there has been poor correlation between the two diseases in humans.38 The answer may be related, in part, to differences in the circumstances under which the two conditions become clinically apparent. In the case of aortic stenosis, excessive valve rigidity, through a combination of leaflet calcification and fibrosis, results in constriction of the valve orifice and the development of symptoms. In contrast, the acute clinical syndromes of atherosclerosis, such as acute myocardial infarction or unstable angina, typically occur when lipid deposition or macrophage infiltration weakens the plaque and results in plaque rupture.39404142 Thus, although both aortic stenosis and atherosclerosis may develop as a result of chronic inflammation, the first is clinically manifest when the response to inflammation causes excessive valve rigidity, whereas the second becomes clinically apparent when the inflammatory response weakens the plaque. Thus, individuals may have differences in susceptibility to the two conditions, depending on whether their calcific and/or fibrotic response to inflammatory injury is vigorous or weak.

In summary, osteopontin, a protein implicated in normal and dystrophic calcification in a variety of tissues, is present in the developing and advanced lesions of aortic stenosis. Further, highly statistically significant associations were demonstrated between the amount of osteopontin protein present and both the degree of calcification and the accumulation of macrophages. Finally, in situ hybridization studies confirmed that a subset of lesion macrophages were actively synthesizing osteopontin. These results are consistent with the hypotheses that, rather than representing a degenerative and unmodifiable process, calcification in valvular aortic stenosis is, at least in part, an active process in which inflammatory cells may participate. Further, the results are consistent with the possibility that valvular aortic stenosis is preventable, either by modification of the inflammatory process or by alteration of expression of proteins, such as osteopontin, which may modulate calcium deposition in this tissue.

Acknowledgments

This work was supported, in part, by a Grant-in-Aid (91-007520) from the American Heart Association to Dr Otto, by a Grant-in-Aid from the American Heart Association, Washington Affiliate, to Dr O’Brien (94-WA-518R), and by grants from the NHLBI to Dr Alpers (HL-42270 and HL-47151). Dr O’Brien is the recipient of a Clinical Investigator Development Award from the NHLBI (HL-02788). The authors thank Lisa Anne Billings for assistance in preparing the manuscript and Winnie Chiu, Susan Rozell, and Kay Gurley for technical assistance.